Systems and assays for identification of compounds that can be used to treat polycystic kidney disease (PKD) are provided. Nematode orthologs of genes involved in PKD are identified and associated with mating behaviors. In particular, nematodes, such as Caenorhabditis elegans, that express mutant and wild-type orthologs of human genes involved in this disease, are used to study the functions of the proteins encoded by the genes, to screen for other genes involved in the disease, to identify mutations involved in the disease, and to screen for drugs that affect PKD. Hence an animal model is provided that permits study of the etiology of polycystic kidney disease and provides a tool to identify the genes and factors involved in the disease pathway, and to identify compounds that may be used to treat or alter the disease progression, lessen its severity or ameliorate symptoms.
Polycystic Kidney Diseases
Polycystic kidney diseases (PKD) are a group of disorders characterized by the presence of a large number of fluid-filled cysts throughout grossly enlarged kidneys (Gabow et al. (1992) Diseases of the Kidney, Schrier et al. eds.). In humans, PKDs can be inherited in autosomal dominant (ADPKD) or autosomal recessive (ARPKD) forms. ADPKD is the more common form and is the most common, dominantly-inherited kidney disease in humans, occurring at a frequency of about 1 in 800. ARPKD occurs at a frequency of about 1 in 10,000.
ADPKD is the most common single-gene disorder leading to kidney failure (see, Emmons et al. (1999) Nature 401:339-340). Since ADPKD is inherited as an autosomal dominant disorder, children of affected parents have a one in two chance of inheriting the disease. Although the kidney is the most severely affected organ, the disease is systemic and affects the liver, pancreas cardiovascular system and cerebro-vascular system. The major manifestation of the disorder is the progressive cystic dilation of renal tubules (Gabow (1990) Am. J. Kidney Dis. 16:403-413), leading to renal failure in half of affected individuals by age 50. Microdissection, histochemical and immunologic studies show that cysts in ARPKD kidneys arise from focal dilations of medullary collecting ducts (McDonald (1991) Semin. Nephrol. 11:632-642). Although end-stage renal failure usually supervenes in middle age (ADPKD is sometimes called adult polycystic kidney disease), children may occasionally have severe renal cystic disease.
ADPKD-associated renal cysts may enlarge to contain several liters of fluid and the kidneys usually enlarge progressively causing pain. Other abnormalities such as hematuria, renal and urinary infection, renal tumors, salt and water imbalance and hypertension frequently result from the renal defect. Cystic abnormalities in other organs, including the liver, pancreas, spleen and ovaries are commonly found in ADPKD. Massive liver enlargement can causes portal hypertension and hepatic failure. Cardiac valve abnormalities and an increased frequency of subarachnoid and other intracranial hemorrhage have also been observed in ADPKD. Progressive renal failure causes death in many ADPKD patients and dialysis and transplantation are frequently required to maintain life in these patients.
Numerous biochemical abnormalities associated with this disease also are observed. These include defects in protein sorting, the distribution of cell membrane markers within renal epithelial cells, extracellular matrix, ion transport, epithelial cell turnover, and epithelial cell proliferation.
Three distinct loci have been shown to cause phenotypically indistinct forms of the AKPKD in humans. These include polycystin-1 (PKD1) on chromosome 16, polycystin-2 (PKD2) on chromosome 4, and polycystin-3 (PKD3) (see, e.g., Reeders et al. (1985) Nature 317:542-544; Kimberling et al. (1993) Genomics 18:467-472; Daoust et al. (1995) Genomics, 25:733-736). The ARPKD mutation is on human chromosome 6 (Zerres et al. (1993) Nature Genet. 7:429-432). Two proteins polycystin-1 (PKD1) and polycystin-2 (PKD2) are defective in human autosomal dominant polycystic kidney disease.
Mutations in either PKD1 or PKD2 cause almost indistinguishable clinical symptoms. Mutations in PKD1 or PKD2 account for 95% of autosomal dominant polycystic disease (Torres et al. (1998) Current Opinion in Nephrology and Hypertension 7:159-169) with greater than 85-90% of disease incidence being due to mutations in PKD1.
The human PKD1 protein is an approximately 4,300 amino-acid integral-membrane glycoprotein with a large amino-terminal extracellular domain and a small, carboxy-terminal cytoplasmic tail. The human PKD1 gene (see, e.g., U.S. Pat. No. 5,891,628), including the complete nucleotide sequence of the gene""s coding region (se SEQ ID No. 1) and encoded amino acid sequence, is known (see, SEQ ID No. 2). The predicted structure of the domains suggested that it is involved in cell-cell interactions or in interactions with the extracellular matrix. The PKD2 protein has similarities to PKD1, but its topology and domain structure suggest that it might act as a subunit of a cation channel. These proteins have been shown to interact directly (Mochizuki et al. (1996) Science 272:1339-1342, Qian (1997) Nature Genetics 16:179-183).
Although these genes have been implicated in the disorders their role in it etiology is not established. In addition, while studies of kidneys from ADPKD patients exhibit a number of different biochemical, structural and physiological abnormalities, the disorder""s underlying causative biochemical defect is not known. Hence the molecular mechanisms leading to cyst enlargement and progressive loss of renal function in the PKDs are not understood. Presently there are no cures or effective treatments, other than palliative treatments, for these diseases. Hence there is a need to understand the underlying biochemistry and physiology of the ADPKD and to provide treatments.
Therefore, it is an object herein to provide a means to identify the underlying biochemistry and genetics of these diseases and to provide a means to identify compounds for use in treatment of these diseases.
Isolated genes, cDNA and encoded proteins from nematodes that participate in a pathway leading to an observable phenotype are provided. In particular, it is shown herein, that a mutation in C. elegans, which gives rise to males that are defective in certain aspects of mating behavior, lies in a gene designed herein lov-1 (location of vulva), and that this gene is an ortholog of the mammalian, particularly human, PKD1 gene. A mutation in a gene designated pkd-2 herein also gives rise to these behaviors. This gene is shown to be an ortholog of the mammalian, including human, PKD2 gene.
The expression pattern of lov-1 and pkd-2 was studied and it was found that promoter sequences of both genes cause reporter genes to be expressed in the rays and the hook sensory neurons required for xe2x80x98responsexe2x80x9d and vulva location. Thus showing that the LOV-1 and PKD-2 proteins are involved in chemosensory or mechanosensory signal transduction in sensory neurons.
Hence genes that are components of a pathway in nematodes are provided and are shown to be linked to observable behaviors. Each of the encoded proteins, LOV-1 and PKD-2 are components in a pathway, which appears to be a signal transduction pathway, that leads to the observed phenotype. The genes from the nematode Caenorhabditis elegans are exemplified herein.
The pathway is shown to be homologous to the pathway in which the human polycystins, PKD1 and PKD2, participate. In particular, it is shown herein, that a mutation in nematodes, which gives rise to males that are defective in mating behavior, lies in a gene designated herein lov-1 (location of vulva). This gene, lov-1, is shown herein to be required for two male sensory behaviors, xe2x80x98responsexe2x80x99 and xe2x80x98location of vulvaxe2x80x99 (Lov).
A second gene, designated pkd-2, that affects this behavior in a similar manner is also identified and provided herein. The encoded proteins are also provided. The gene, cDNA, and encoded protein is also provided. In an exemplary embodiment, the C. elegans genome sequence was used to isolate pkd-2. This gene is a nematode ortholog of the mammalian, particularly human PKD2 gene. Strains that contain knock-out mutants of this gene also exhibit the defective mating behaviors.
In an exemplary embodiment, provided herein are the C. elegans genes, designated lov-1 and pkd-2. SEQ ID No. 3 sets forth the complement (i.e., the non-coding strand) of the lov-1 gene from C. elegans. SEQ ID No. 4 sets forth the sequence of amino acids of the protein (N-terminus to C-terminus)). SEQ ID No. 5 sets forth the complement (i.e., the non-coding strand) of the C. elegans pkd-2 gene from C. elegans. SEQ ID No. 6 sets forth the encoded sequence of amino acids.
Also provided are the mutants of the genes, lov-1, and pkd-2 and the resulting mutant encoded proteins. Nucleic acid molecules encoding mutants of these genes are also provided. For example, deletion mutants of these genes, particularly deletion mutants that substantially or completely knock-out gene product function, are provided. Thus, nucleic acid molecules containing deletions of each of these genes and deletion mutants that alter the phenotype of nematodes, such as C. elegans, that contain these mutant genes are also provided. Constructs, vectors, plasmids and strains containing each of the nucleic molecules are also provided. Also provided are strains defective in these genes.
Also provided are strains containing the mutant nucleic acids. Strains that manifest the defective male sensory behaviors are also provided herein. Constructs containing the genes, vectors containing the constructs, cells containing the vectors and transgenic C. elegans. Assays that use these strains of C. elegans are also provided.
As noted, it is shown herein that these genes are human homologs of the human genes that encode polycystins, proteins polycystin-1 (PKD1) and polycystin-2 (PKD2), which are defective in human autosomal dominant polycystic kidney disease. Hence, the genes and nematode strains provide model systems for studying this pathway, identifying additional components of the pathway, and for use in drug screening assays to identify compounds affect the pathway and/or compounds that serve as leads for development of drugs for treatment of polycystic kidney disease.
Each gene is shown to affect two sensory behaviors in C. elegans. One behavior designated xe2x80x9cResponsexe2x80x9d and refers to the response of males to hermaphrodites; and the other behavior, designated xe2x80x9cLovxe2x80x9d refers to location of the vulva by the male. Strains that are defective in either or both of these genes are also provided. In particular deletion mutants are provided.
By correlating the phenotypic behaviors with wild-type or defects in these genes, nematodes, such as C. elegans, can be used to identify other genes involved in this pathway and also means for direct screening for lead candidate compounds for drugs for treatment of PKD. Identification of additional genes necessary for PKD function can provide additional diagnostic tools for PKD. Hence, provided herein are mutant strains of C. elegans and assays that use the strains.
Also provided herein are assays that employ the constructs, vectors, plasmids and strains containing each of the nucleic molecules are also provided. In particular, in one type of assays wild-type nematodes are mutagenized or treated with a test compound, and those that exhibit a change in behavior are identified.
In other types of assays, nematodes that are defective in LOV and/or Response are mutagenized or treated with a compound, and those that exhibit a change in behavior are identified. Test compounds or mutations responsible for the change in behavior are identified. Such compounds are candidates for treatment of PKDs.
Among these methods are those that involved contacting a nematode that exhibits normal mating behavior with a test compound; and selecting compounds that result in altered mating behavior, wherein the altered mating behavior comprises alteration in the behavior involving location of vulva and/or response to contact with the hermaphrodite.
Also provided are methods for identifying genes involved in autosomal dominant polycystic kidney disease (ADPKD). Among these methods are those in involving mutagenizing nematodes that exhibit normal mating behavior; and identifying and selecting nematodes that exhibit altered mating behavior, where the altered mating behavior is manifested as an alteration in location of vulva and/or response to contact with the hermaphrodite. The mutated gene(s) responsible for the alteration in behavior are then identified. Databases or libraries of mammalian genes can be screened to identify homologs of these genes, which can then serve as therapeutic or diagnostic targets or aid in elucidation of the disease pathology.
Methods for identifying compounds that are candidate therapeutic agents for treatment of autosomal dominant polycystic kidney disease (ADPKD) are provided. Among the methods are those in which normal males are treated with a candidate compound. Compounds that result in changes in mating behaviors or changes in mating efficiencies are selected.
Methods for identifying genes involved in the disease pathway are also provided. Among the methods are those in which normal males are mutagenized. Offspring that exhibit changes in mating behaviors or changes in mating efficiencies are selected and mutated genes are identified and shown to be part of the pathway. Mammalian, particularly human, homologs of the mutated genes are then identified. Such genes are likely to be part of the disease pathway. Such genes can serve as therapeutic targets and disease markers for diagnostic.
Other assays use nematode strains that have mutations in either or both of lov-1 or pkd-2. As described herein, suppressor and enhancer genetics can be used to assign functions to genes, to assign genes to pathways, to identify the key switches in these pathways and to provide a sensitive assay to identify new genes in a pathway and lead compounds that modulate the activity of genes and/or gene products in the pathway.
Assays that identify the role of PKD proteins in sensory function are also provided. Since lov-1 and pkd-2 are expressed in CEM neurons, they have activity in other sensory functions, such as finding the mating partner at a distance. Accordingly assays using sexual chemotaxis or kinesis are provided. For example, males that are mutagenized or treated with a test compound are placed on a surface containing males and hermaphrodites, and are then observed to assess whether they can choose between males and hermaphrodites. If the male is defective in this sensory function, it will not distinguish between males and hermaphrodites.
Assays that use dominant negative forms of PKD in nematodes or in other cells to identify mutations and/or compounds that inhibit PKD function are also provided. Transgenic nematodes that express a version of the LOV-1 or PKD-2 protein that inhibits the activity of LOV-1 and/or PKD-2 as assessed by manifestation of the altered LOV and/or response phenotypic behavior(s) are used in these assays. Transgenic nematodes can be produced by any method known to those of skill in the art, including, but are limited to, injection of the nucleic acid into the embryos or cells of the animal. Transgenic nematodes that contain a dominant negative lov-1 or pkd-2 transgene are contacted with a test compound, and compounds that interfere with a remaining activity of the LOV-1 or PKD-2 protein are selected. Alternatively, these transgenic nematodes are mutagenized and mutants that lose a remaining activity are selected and the gene or mutation responsible for the loss or that contributes to the loss is identified.
Assays based on localization and trafficking of LOV-1 and/or PKD-2 within a cell or cells are also provided. These assays can identify regulators and factors necessary for synthesis and transport of LOV-1 and/or PKD-2 proteins and employ strains in which LOV-1 and PKD-2 are expressed linked to a detectable label, such as a fluorescent protein. These strains are used to assess the effects of compounds or mutagenesis on the trafficking patterns of LOV-1 and PKD-2 and cellular location(s) of the proteins in the animal. Identified mutations can be mapped and the genes identified. If mammalian, particularly human, homologs of these identified genes exist, such genes can serve as therapeutic or diagnostic targets and can aid in elucidation of the disease in mammals, particularly humans.
Assays for identification of transcriptional regulators of expression of lov-1 and/or pkd-2 are also provided. These assays screen for loss or alteration of expression of either gene and use transgenic nematodes with a reporter gene, such as a gene encoding a FP or lacZ or other detectable product, linked to the nucleic acid encoding lov-1 or pkd-2. The animal is mutagenized or treated with a test compound and loss of expression or reduction in expression of either gene is assessed. These assays identify regulators of and factors that affect lov-1 and pkd-2 expression. Mammalian, particularly human homologs of these regulators and factors are identified. Such regulators and factors can be therapeutic or diagnostic targets, and/or can aid in developing an understanding of the development and progression of PKD in mammals.
Kits for performing the assays, particularly, the drug screening assays, are also provided. The kits include transgenic or wild-type nematodes or both that express either wild-type or a mutant or a transgenic form of lov-1 and/or pkd-2. The nematodes may be on plates, in wells or in any form suitable for the assays. Kits containing nucleic acid encoding either of the two genes or probes based upon these sequences or reporter gene constructions containing all or portions of either or both genes are also provided. The nucleic acids may be in solution, in lyophilized or other concentrated form, or may be bound to a suitable substrate. The kits can include additional reagents for performing the assays, such reagents include any for performing any of the steps of the methods. The kits include instructions for performing the assays.